Changes in chromatin accessibility are not concordant with transcriptional changes for single-factor perturbations.

A major goal in the field of transcriptional regulation is the mapping of changes in the binding of transcription factors to the resultant changes in gene expression. Recently, methods for measuring chromatin accessibility have enabled us to measure changes in accessibility across the genome, which are thought to correspond to transcription factor-binding events. In concert with RNA-sequencing, these data in principle enable such mappings; however, few studies have looked at their concordance over short-duration treatments with specific perturbations. Here, we used tandem, bulk ATAC-seq, and RNA-seq measurements from MCF-7 breast carcinoma cells to systematically evaluate the concordance between changes in accessibility and changes in expression in response to retinoic acid and TGF-ß. We found two classes of genes whose expression showed a significant change: those that showed some changes in the accessibility of nearby chromatin, and those that showed virtually no change despite strong changes in expression. The peaks associated with genes in the former group had lower baseline accessibility prior to exposure to signal. Focusing the analysis specifically on peaks with motifs for transcription factors associated with retinoic acid and TGF-ß signaling did not reduce the lack of correspondence. Analysis of paired chromatin accessibility and gene expression data from distinct paths along the hematopoietic differentiation trajectory showed a much stronger correspondence, suggesting that the multifactorial biological processes associated with differentiation may lead to changes in chromatin accessibility that reflect rather than driving altered transcriptional status. Together, these results show many gene expression changes can happen independently of changes in the accessibility of local chromatin in the context of a single-factor perturbation.

Gene regulation gravitates toward either addition or multiplication when combining the effects of two signals.

Two different cell signals often affect transcription of the same gene. In such cases, it is natural to ask how the combined transcriptional response compares to the individual responses. The most commonly used mechanistic models predict additive or multiplicative combined responses, but a systematic genome-wide evaluation of these predictions is not available. Here, we analyzed the transcriptional response of human MCF-7 cells to retinoic acid and TGF-ß, applied individually and in combination. The combined transcriptional responses of induced genes exhibited a range of behaviors, but clearly favored both additive and multiplicative outcomes. We performed paired chromatin accessibility measurements and found that increases in accessibility were largely additive. There was some association between super-additivity of accessibility and multiplicative or super-multiplicative combined transcriptional responses, while sub-additivity of accessibility associated with additive transcriptional responses. Our findings suggest that mechanistic models of combined transcriptional regulation must be able to reproduce a range of behaviors.

Gene Networks with Transcriptional Bursting Recapitulate Rare Transient Coordinated High Expression States in Cancer.

Non-genetic transcriptional variability is a potential mechanism for therapy resistance in melanoma. Specifically, rare subpopulations of cells occupy a transient pre-resistant state characterized by coordinated high expression of several genes and survive therapy. How might these rare states arise and disappear within the population? It is unclear whether the canonical models of probabilistic transcriptional pulsing can explain this behavior, or if it requires special, hitherto unidentified mechanisms. We show that a minimal model of transcriptional bursting and gene interactions can give rise to rare coordinated high expression states. These states occur more frequently in networks with low connectivity and depend on three parameters. While entry into these states is initiated by a long transcriptional burst that also triggers entry of other genes, the exit occurs through independent inactivation of individual genes. Together, we demonstrate that established principles of gene regulation are sufficient to describe this behavior and argue for its more general existence. A record of this paper's transparent peer review process is included in the Supplemental Information.

Enrichment of Targetable Mutations in the Relapsed Neuroblastoma Genome.

Neuroblastoma is characterized by a relative paucity of recurrent somatic mutations at diagnosis. However, recent studies have shown that the mutational burden increases at relapse, likely as a result of clonal evolution of mutation-carrying cells during primary treatment. To inform the development of personalized therapies, we sought to further define the frequency of potentially actionable mutations in neuroblastoma, both at diagnosis and after chemotherapy. We performed a retrospective study to determine mutation frequency, the only inclusion criterion being availability of cancer gene panel sequencing data from Foundation Medicine. We analyzed 151 neuroblastoma tumor samples: 44 obtained at diagnosis, 42 at second look surgery or biopsy for stable disease after chemotherapy, and 59 at relapse (6 were obtained at unknown time points). Nine patients had multiple tumor biopsies. ALK was the most commonly mutated gene in this cohort, and we observed a higher frequency of suspected oncogenic ALK mutations in relapsed disease than at diagnosis. Patients with relapsed disease had, on average, a greater number of mutations reported to be recurrent in cancer, and a greater number of mutations in genes that are potentially targetable with available therapeutics. We also observed an enrichment of reported recurrent RAS/MAPK pathway mutations in tumors obtained after chemotherapy. Our data support recent evidence suggesting that neuroblastomas undergo substantial mutational evolution during therapy, and that relapsed disease is more likely to be driven by a targetable oncogenic pathway, highlighting that it is critical to base treatment decisions on the molecular profile of the tumor at the time of treatment. However, it will be necessary to conduct prospective clinical trials that match sequencing results to targeted therapeutic intervention to determine if cancer genomic profiling improves patient outcomes.

Antitumor Response of VEGFR2- and VEGFR3-Amplified Angiosarcoma to Pazopanib.

BACKGROUND: Angiosarcoma is a malignant neoplastic disease originating from or differentiating toward vascular endothelium, for which systemic pharmacologic treatment has limited durability. The molecular oncogenesis of angiosarcoma is often linked to inappropriate activations of vascular endothelial growth factor receptor (VEGFR) family members, which presents an opportunity for the use of therapy that selectively targets the machinery of vascular signaling. METHODS: Hybridization capture of 3,320 exons of 182 cancer-related genes and the introns of 14 genes frequently rearranged in cancer was applied to more than 50 ng of DNA extracted from a formalin-fixed, paraffin-embedded biopsy of recurrent angiosarcoma and was sequenced to high, uniform coverage of 939x. RESULTS: The angiosarcoma harbored amplifications of VEGFR2 (KDR) of 8 copies and VEGFR3 (FLT4) of 16 copies. The patient was initially treated with sorafenib, an inhibitor of VEGFR2, and developed progressive disease. The patient then received pazopanib, an inhibitor of VEGFR2 and VEGFR3 and experienced a potent antitumor response resulting in clinically stable disease for 6 months. CONCLUSIONS: This exceptional response to pazopanib treatment suggests that a subset of patients with angiosarcoma with genomic alterations in vascular signaling genes may respond well to pazopanib.

Identification and characterization of RET fusions in advanced colorectal cancer.

There is an unmet clinical need for molecularly directed therapies available for metastatic colorectal cancer. Comprehensive genomic profiling has the potential to identify actionable genomic alterations in colorectal cancer. Through comprehensive genomic profiling we prospectively identified 6 RET fusion kinases, including two novel fusions of CCDC6-RET and NCOA4-RET, in metastatic colorectal cancer (CRC) patients. RET fusion kinases represent a novel class of oncogenic driver in CRC and occurred at a 0.2% frequency without concurrent driver mutations, including KRAS, NRAS, BRAF, PIK3CA or other fusion tyrosine kinases. Multiple RET kinase inhibitors were cytotoxic to RET fusion kinase positive cancer cells and not RET fusion kinase negative CRC cells. The presence of a RET fusion kinase may identify a subset of metastatic CRC patients with a high response rate to RET kinase inhibition. This is the first characterization of RET fusions in CRC patients and highlights the therapeutic significance of prospective comprehensive genomic profiling in advanced CRC.

Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors.

UNLABELLED: Focal amplification and activating point mutation of the MET gene are well-characterized oncogenic drivers that confer susceptibility to targeted MET inhibitors. Recurrent somatic splice site alterations at MET exon 14 (METex14) that result in exon skipping and MET activation have been characterized, but their full diversity and prevalence across tumor types are unknown. Here, we report analysis of tumor genomic profiles from 38,028 patients to identify 221 cases with METex14 mutations (0.6%), including 126 distinct sequence variants. METex14 mutations are detected most frequently in lung adenocarcinoma (3%), but also frequently in other lung neoplasms (2.3%), brain glioma (0.4%), and tumors of unknown primary origin (0.4%). Further in vitro studies demonstrate sensitivity to MET inhibitors in cells harboring METex14 alterations. We also report three new patient cases with METex14 alterations in lung or histiocytic sarcoma tumors that showed durable response to two different MET-targeted therapies. The diversity of METex14 mutations indicates that diagnostic testing via comprehensive genomic profiling is necessary for detection in a clinical setting. SIGNIFICANCE: Here we report the identification of diverse exon 14 splice site alterations in MET that result in constitutive activity of this receptor and oncogenic transformation in vitro. Patients whose tumors harbored these alterations derived meaningful clinical benefit from MET inhibitors. Collectively, these data support the role of METex14 alterations as drivers of tumorigenesis, and identify a unique subset of patients likely to derive benefit from MET inhibitors.

The Clinical Use of Genomic Profiling to Distinguish Intrapulmonary Metastases From Synchronous Primaries in Non-Small-Cell Lung Cancer: A Mini-Review.

The ability to reliably distinguish synchronous primary non-small-cell lung cancer (NSCLC) from intrapulmonary metastatic spread affects staging and treatment decisions in resected NSCLC. Adjuvant therapy for early-stage NSCLC is complicated and recommendations are primarily based on older data from trials that used now-outdated staging systems. Patients found to have 2 tumors with similar morphology in the same lobe are currently staged as pathologic T3 (pT3) but such cases represent a minority of patients in adjuvant lung cancer trials. Potentially more precise than tumor morphology alone, comprehensive genomic profiling technologies have the power to discriminate whether tumors in the same lobe represent 2 separate primary lesions or localized spread of a single lesion. In addition to lineage insights, tumor profiling simultaneously provides information on actionable genomic alterations. In this review we discuss the data that support the ability of molecular technologies to distinguish synchronous primary tumors from intrapulmonary metastases and discuss the use of molecular assays as an adjunct to current staging systems. Two cases are presented to highlight the potential immediate clinical implications of comprehensive genomic profiling.

Prospective comprehensive genomic profiling of advanced gastric carcinoma cases reveals frequent clinically relevant genomic alterations and new routes for targeted therapies.

BACKGROUND: Gastric cancer (GC) is a major global cancer burden and the second most common cause of global cancer-related deaths. The addition of anti-ERBB2 (HER2) targeted therapy to chemotherapy improves survival for ERBB2-amplified advanced GC patients; however, the majority of GC patients do not harbor this alteration and thus cannot benefit from targeted therapy under current practice paradigms. MATERIALS AND METHODS: Prospective comprehensive genomic profiling of 116 predominantly locally advanced or metastatic (90.0%) gastric cancer cases was performed to identify genomic alterations (GAs) associated with a potential response to targeted therapies approved by the U.S. Food and Drug Administration or targeted therapy-based clinical trials. RESULTS: Overall, 78% of GC cases harbored one clinically relevant GA or more, with the most frequent alterations being found in TP53 (50%), ARID1A (24%), KRAS (16%), CDH1 (15%), CDKN2A (14%), CCND1 (9.5%), ERBB2 (8.5%), PIK3CA (8.6%), MLL2 (6.9%), FGFR2 (6.0%), and MET (6.0%). Receptor tyrosine kinase genomic alterations were detected in 20.6% of cases, primarily ERBB2, FGFR2, and MET amplification, with ERBB2 alterations evenly split between amplifications and base substitutions. Rare BRAF mutations (2.6%) were also observed. One MET-amplified GC patient responded for 5 months to crizotinib, a multitargeted ALK/ROS1/MET inhibitor. CONCLUSION: Comprehensive genomic profiling of GC identifies clinically relevant GAs that suggest benefit from targeted therapy including MET-amplified GC and ERBB2 base substitutions.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing.

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.